Mesenchymal stem cells give rise to the progenitors of many differentiated phenotypes including osteocytes, chrondocytes, myocytes, adipocytes, fibroblasts, and marrow stromal cells. Certain mesenchymal progenitor cells are capable of self-renewal and undergo expansion in the presence of transforming growth factor .beta.1 (TGF.beta.1), a pleotropic cytokine with autocrine and paracrine functions.
Transforming growth factor .beta. (TGF.beta.), a 25 KDa peptide found abundantly in platelets and bone, released in response to tissue injury, is becoming an increasingly important tool for immunomodulation, wound healing, and tissue repair. TGF.beta. is also a chemoattractant for cells of mesenchymal origin, and as such, recruits fibroblasts to the site of injury, stimulates angiogenesis and de novo synthesis of extracellular matrix proteins in concert with the up-regulation of inhibitors of matrix degradation. See Roberts A. B., Sporn M. B.: The Transforming Growth Factor-.beta.s, pp. 420-472 (1990). TGF.beta.1 and TGF.beta.2 are potent immunoregulatory agents, suppressing the proliferation and function of T and B lymphocytes in vitro (Id.) and in vivo (See Wrann M, et al., "T Cell Suppressor Factor from Human Glioblastoma Cells Is a 12.5 KD Protein Closely Relating to Transforming Growth Factor-beta," EMP. J. 6:1633-36 (1987)). Hence, TGF.beta. appears to play a crucial role in clinically relevant disorders of immune surveillance, tissue regeneration, and repair. Moreover, repair after tissue injury such as burns, myocardial infarction, cerebral ischemia and trauma, as well as surgical wound healing, may be accelerated by a single systemic infusion or local application of this peptide growth factor. See Beck S. L., et al. "TGF-.beta.1 Induces Bone Closure of Skull Defects," J. Bone Mineral Res. 6(1991). The therapeutic effects of TGF.beta. administration may be augmented and/or prolonged by its pronounced autocrine and paracrine functions.
U.S. Pat. No. 5,486,359 (Caplan et al.) is directed to isolated human mesenchymal stem cells, and a method for their isolation, purification, and culturing. Caplan et al. also describe methods for characterizing and using the purified mesenchymal stem cells for research diagnostic and therapeutic purposes.
Heretofore, TGF.beta.1 has not been demonstrated to function as a survival factor, as opposed to a growth (proliferation) factor.
SUMMARY OF THE INVENTION
The present invention is directed to a method for expressing a recombinant protein from bone-marrow derived cells, comprising the steps of treating the bone marrow-derived cells in vitro with a TGF.beta.1 protein, which selects from the cells a population of cells that are responsive to the TGF.beta.1 protein. The selected calls can be thereafter expanded in the cell culture, after which the cells can be transduced with a DNA segment encoding a therapeutic protein to cause the cells to express the therapeutic protein. The transduced cells can then be introduced into a recipient to produce a therapeutic result.
Bone marrow-derived cells for use in the present invention include mesenchymal stem cells and differentiated mesenchymal cells, such as stromal cells.
In a preferred embodiment, the TGF.beta.1 protein is a TGF.beta.1 fusion protein comprising an extracellular matrix binding site, which is preferably a collagen binding site. The extracellular matrix binding site of the TGF.beta.1 fusion protein can then be used to target the TGF.beta.1 fusion protein to an extracellular matrix, such as collagen.
In another aspect, the present invention is directed to a gene therapy method comprising the steps of capturing TGF.beta.1-responsive, bone marrow-derived cells under low serum conditions in a collagen matrix impregnated with a TGF.beta.1 fusion protein comprising a von Willebrand's factor-derived collagen binding site (TGF.beta.1-vWF) which targets the TGF.beta.1 fusion protein to the collagen matrix. The captured cells then can be expanded in the cell culture to form differentiated cell colonies. These expanded cell colonies can then be transduced in vitro with a viral vector comprising a gene encoding a therapeutic protein, wherein the gene is expressed to produce the therapeutic protein. The transduced cells can thereafter be introduced into a mammal, such as a human, to produce a therapeutic result.
In a particular embodiment of the invention, it has been discovered that mesenchymal progenitor cells isolated with a TGF.beta.1-vWF fusion protein, expanded in culture, and transduced with a retroviral vector containing the gene encoding factor IX expressed significant levels of factor IX protein. Moreover, when the transduced cells were transplanted into immunocompetent mice, the human factor IX transgene was expressed in vivo.